Rabbit anti tnf α

Protein from spinal cord tissues and PC12 cells was extracted in an NP‐40 lysis buffer. An equal amount of protein was fractionated by 11.5% SDS‐PAGE, and transferred onto PVDF membranes (Bio‐Rad Laboratories, Hercules, CA, USA). Membranes were blocked with 5% freshly prepared milk‐TBST for 90 min. at room temperature and then incubated overnight at 4°C with primary antibodies (rabbit anti‐cleaved‐caspase 3 (1:1000), goat anti‐Iba‐1 (1:1000), rabbit anti‐TNF‐α (1:1000), mouse anti‐TLR4 (1:10,000), goat anti‐IL‐6 (1:300) or mouse anti‐GAPDH (1:1000) from Santa Cruz Biotechnology; mouse anti‐NF‐κB (1:400), rabbit anti‐IκB (1:400), mouse anti‐p‐IκB (1:400) from Abcam). After being washed in TBST, membranes were incubated with appropriate secondary antibodies for 1 hr at room temperature. Proteins were detected using an enhanced chemiluminescence (ECL) kit (Bio‐Rad). Signal intensities were quantified by densitometry using Image Lab 3.0 software (Bio‐Rad). Data were normalized to total or loading controls 23, 24.

Four series of sections were deparaffinized in xylene and rehydrated. Endogenous peroxidase activity was extinguished with 10% H₂O₂ and heat-induced antigen retrieval was performed using Tris-EDTA buffer (1 mM EDTA solution, 10 mM Tris base, and 0.05% Tween 20 in distilled water, pH 9). Sections were incubated overnight in a buffer solution (5% skim milk, 0.1% Tween 20 in Tris-buffered saline) containing one of the following primary antibodies: rabbit anti-TNF-α, rabbit anti-iba1, goat anti-caspase 3, and goat anti-Fas (1 : 100, Santa Cruz Biotechnology, CA). Then, sections were incubated in an HRP-conjugated secondary antibody and processed according to the instructions of the kit manufacturer (ABCAM, Cambridge, UK). Finally, they were stained with chromogen substrate 3,3′-diaminobenzidine hydrochloride (DAB, ABCAM) (10 min), counterstained with hematoxylin (ABCAM) (5 min), dehydrated through a graded ethanol series, cleared in xylene, and covered with a glass coverslip. Tris-TBS buffer (0.1% Tween 20 in Tris-buffered saline, pH = 7.6) was used for interstep rinsing, as recommended by the kit manufacturer (ABCAM). Expressions of these markers of inflammation (Tumor Necrosis Factor-alpha/TNF-α, iba1), inflammation-related cell death (Fas receptor), and apoptosis (caspase-3) were observed using a light microscope under 20x, 40x, and 120x objectives.